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Abstract

Background

Probiotics are used to provide health benefits. The present study tested the effect
of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a
group of young healthy women eating a defined diet.

Findings

26 women aged 18-21 (median 19) years residing in a hostel were given 200 ml normal
yoghurt every day for a week, followed by probiotic yoghurt containing Bifidobacterium lactis Bb12® (109 in 200 ml) for three weeks, followed again by normal yoghurt for four weeks. Stool
samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human
beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt
well. Human beta-defensin-2 levels in faeces were not altered during the course of
the study. On the other hand, compared to the basal sample, faecal IgA increased during
probiotic feeding (P = 0.0184) and returned to normal after cessation of probiotic
yoghurt intake.

Conclusions

Keywords:

Probiotics; innate immunity; health promotion; mucosal defences

Introduction

Probiotic foods are widely used to promote health. They are also sometimes used to
prevent or treat specific gastrointestinal illnesses. Recent studies have shown that
ingestion of foods containing probiotic Lactobacillus or Bifidobacterium strains prevent or reduce morbidity from enteric infections and lower respiratory
tract infections [1,2]. Bifidobacterium lactis strain Bb12® is a probiotic microbe that is widely consumed in the form of probiotic yoghurt. Probiotic
yoghurt containing this microbe is reported to have beneficial effects on metabolism
including lowered serum LDL-cholesterol in patients with type 2 diabetes,[3] increased HDL cholesterol in adult women [4] and improved glucose tolerance during pregnancy [5,6]. Bb12® administration has also been shown to increase faecal secretory IgA excretion in preterm
infants [7]. The health claims of probiotics have been demonstrated with varying levels of evidence,
with only a few being substantiated using double blind randomized controlled trials.

Dietary practices in India are different from that in the developed countries where
evidence of probiotic efficacy has been gathered. This, together with the occurrence
of frequent gastrointestinal infections in childhood and the widespread consumption
of home-made yoghurt in the diet, may result in differences in the gastrointestinal
response to probiotic bacteria in Indians. The present study evaluated the effect
of daily ingestion of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of IgA and β-defensin 2 in healthy adult southern Indian women
volunteers.

Methods

Participants and Interventions

Healthy young adult women living in a hostel and eating food prepared in the hostel
kitchen were recruited for the study. Individuals who had received a course of antibiotics
within the last month were excluded as were those who intended to travel out of the
city during the course of the feeding trial. Participants were briefed about the nature
and purpose of the study, the importance of compliance with the study intervention,
and the importance of maintaining a daily record of bowel movements and any abdominal
symptoms. Volunteers were given a small monetary incentive to participate in the study.
All participants received normal yoghurt daily for the first week of the feeding study,
following which they received probiotic yoghurt daily for the next three weeks. This
was again followed by regular yoghurt feeding for the next four weeks. Normal yoghurt
was prepared in the diet kitchen by boiling standardized toned milk (3.0% fat & 8.5%
msnf) and then cooling to 40°C, following which starter culture (YCX-11, Chr Hansen)
was added at 1 unit per 10 litres of milk. The milk was distributed in 200 ml cups
which were incubated at 40°C until the pH reached 4.6, and cups then transferred to
a refrigerator for cooling. Probiotic yoghurt was prepared by adding Bb-12® (Batch no 2927446, Chr Hansen) at a concentration of 0.0006% to the cultured milk
prepared as above. This dosage was calculated to provide approximately 109 cfu of Bifidobacterium per 200 ml serving of yoghurt. The investigator responsible for providing the diluted
starter culture did not participate in yoghurt distribution or in the laboratory analyses.
Bifidobacterial concentrations in yoghurt were checked by culture of diluted yoghurt
(1:10 in peptone water broth, 0.9% NaCl, 0.85% peptone) and serial dilutions (10-2 to 10-10) were made and plated on Reinforced Clostridial Agar (13.5 g/250 ml, pH 6.8) (Himedia
laboratories, Mumbai, India, Catalog number-M154-500G) containing mupirocin (25 μg/L
of medium) (RM-6090, Himedia laboratories, Mumbai, India). Plates were incubated at
37°C in anaerobic jars for three days and colony counts calculated from the growth
in serial dilutions.

Yoghurt was prepared fresh every morning and distributed at lunch time to the participants.
As all participants had lunch in the hostel mess, this allowed distribution at a single
point and consumption of yoghurt under supervision. The study was preceded by focus
group discussions. Participants were interviewed by a social worker and a dietician.
Demographic data were recorded and socioeconomic score was calculated [8]. A 24 hour dietary recall, together with a food frequency questionnaire of commonly
used foods over the past three months, was used to calculate nutrient intake using
standard tables of the composition and nutritive value of Indian foods [9]. Standard cups and spoons were used to measure meal sizes. Each participant was given
a printed study diary and asked to record on a daily basis the following - stool frequency,
stool consistency, abdominal pain, gaseousness, and any other symptoms that the participant
noted during this period. Each participant was requested to give a fresh sample of
stool before the study commenced, at the end of the first intervention period (i.e.
at 4 weeks) and at the end of the second intervention period (i.e. at 8 weeks). The
study protocol, incentive, and consent forms were approved by the Institutional Review
Board. All participants provided written consent.

Fresh samples of stool were collected in the morning and transported immediately to
the laboratory and stored at -80°C. Secretory IgA and beta-defensin 2 were assayed
by ELISA (K8870 and K6500, Immunodiagnostik, Germany) following manufacturer's instructions.
Stool samples weighing 80-120 mg were diluted with appropriate amounts of buffer provided
in the kit to give constant dilutions, vortexed, and centrifuged at 13000 rpm for
5 minutes in 1.5 ml tubes. Supernatants were diluted 1:250 in wash buffer. Standards,
controls and stool samples were simultaneously run in the ELISA and the concentration
of secretory IgA and β-defensin 2 were read against a standard curve and expressed
as concentration per gram wet weight of stool.

Statistics

Values are presented as numbers for categorical variables and as median with interquartile
range for continuous data. Analysis of significance of differences between groups
was done using paired t tests comparing each group with the others.

Results

26 women of median age 19 (range 18-21) years were recruited for the study. Their
median (range) socioeconomic score was 16 (13-25). A score of 11-15 is categorized
as middle class and 16-25 as upper middle class). Their median (range) height was
154 (150-171) cm, weight was 53.5 (43-77) kg, and BMI was 22.1. (18.5-26.3). The dietary
intakes and preferences were as follows. Eight were lacto-vegetarian, while the rest
ate meat at least once a week. Their median (range) daily nutrient intake was energy
1425 (1025-1735) kcal, protein 30 (20-40) g, fat 19 (16-26) g, simple carbohydrates
17 (14-45) and complex carbohydrates 246 (143-336).

No significant change in bowel frequency (median daily frequency 1) or stool consistency
was reported during consumption of the probiotic yoghurt. There was no significant
complaint of abdominal pain or bloating or other abdominal symptoms during probiotic
yoghurt feeding compared to normal yoghurt feeding. Two volunteers each had abdominal
pain during probiotic yoghurt feeding and normal yoghurt feeding. 13 and 8 participants
respectively had symptoms consistent with a common cold during three weeks of probiotic
or normal yoghurt feeding.

Figure 1 shows the β-defensin 2 concentrations in faeces of volunteers fed probiotic yoghurt
or normal yoghurt. There was no change in the concentration of this immune effector
molecule brought about by the ingestion of probiotics. On the other hand, faecal levels
of secretory IgA were significantly higher in the participants during the probiotic
yoghurt feeding period compared to basal periods (P = 0.0184). Faecal IgA levels after
cessation of probiotic yoghurt feeding showed a trend towards being increased compared
to the baseline levels (P = 0.0822) and was not significantly different from the peak
levels observed during probiotic yoghurt feeding (P = NS) (Figure 1).

Figure 1.Box and whiskers plots showing the effect of probiotic yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory IgA and human beta-defensin-2. The middle line represents the median, the box represents the interquartile range
and the whiskers represent the total range. Faecal IgA excretion significantly increased
during after probiotic yoghurt feeding and returned to normal levels after cessation
of the probiotic.

Discussion

This study in a group of young adult women in south India shows that the probiotic
yoghurt was well tolerated and that faecal excretion of secretory immunoglobulin A
was significantly increased during feeding of probiotic yoghurt compared to the baseline.
Although the levels decreased after probiotic yoghurt was stopped, they did not return
to normal and tended to be higher than baseline levels. This suggests that the effect
of the probiotic yoghurt persisted even after cessation of feeding this; there is
also be a possibility that normal yoghurt contributed to elevated faecal sIgA, that
was more pronounced during feeding of probiotic yoghurt. These results are likely
to be generalizable. Studies in healthy children and preterm infants have shown an
increase in faecal IgA excretion in response to probiotic interventions [7,10]. A study in animals showed that probiotic yoghurt containing Lactobacillus casei
increased IgA secretion in mice [11]. On the other hand, a study in infants did not find any change in faecal IgA after
probiotic administration [12]. In another study, faecal IgA output in pre-term infants was increased in response
to both mother's milk and to fermented formula (in which the probiotic was killed
by heating) [13].

Probiotics are also known to upregulate defensin expression and to increase defensin
secretion from intestinal epithelium. Both lactobacilli and probiotic E. coli have been shown to increase human beta-defensin 2 (hBD-2) secretion from CaCo-2 cells
through stimulation of TLR2 and TLR5 respectively [14-16]. In a feeding trial in healthy adult volunteers, the probiotic E. coli Nissle strain increased fecal hBD-2 excretion in healthy adult volunteers [17]. In the present study, there was no obvious effect on faecal hBD-2 excretion during
the probiotic yoghurt feeding period. Thus far, an effect on human beta-defensin expression
has been noted only with lactobacilli and with E. coli Nissle 2. In a study where
11 lactobacilli were tested, only 2 of the strains increased hBD-2 mRNA expression
in CaCo-2 cells. The difference between lactobacilli strains in their ability to induce
hBD-2 correlated with the presence of genes encoding glycosylated cell surface structures
[18]. Although we did not examine the probiotic used in this study for these genes, this
appears a likely explanation for the absence of an effect on hBD-2 excretion in stool.

This study had several drawbacks. It was a non-randomised study and the participants
could potentially differentiate between the two different kinds of yoghurt on the
basis of smell and taste, although the nature of the two was not known to them. These
were not commercially available standardized products, although we took care to make
them to a standard protocol. The generalizability of these results to other products
and populations is therefore not to be assumed.

The present study appears to be the first report of an effect of probiotic foods on
faecal IgA in healthy adults. Increased IgA output was noted only with yoghurt containing
Bb12® and not with normal yoghurt. An increase in faecal IgA may explain the ability of
probiotics to prevent diarrhoeal illness in specific settings. It is possible that
this may also explain beneficial effects of probiotics in preventing severe lower
respiratory tract infections.

Competing interests

The study was supported by a grant from Chr. Hansen (India) Private Limited. The funder
participated in the design of the study, provided the starter cultures and probiotics,
and was involved in initial training of the dieticians in manufacturing and quality
control of the yoghurt. The funder was not involved in laboratory analyses, data analysis,
data reporting, or write-up of the manuscript.

Authors' contributions

JK and PD were responsible for specimen processing and laboratory analyses; RSD was
responsible for dietary analyses; RSD, NVM and SP were responsible for yoghurt preparation
and quality control; RMR, RV and JM were responsible for recruitment and supervision
of the feeding study; SP was responsible for overall supervision; BSR, RMR, JK and
SP were responsible for design; JK and BSR were responsible for write-up of the manuscript.
All authors read and approved the final manuscript.

Acknowledgements

Jayakanthan Kabeerdoss received a Senior Research Fellowship from the Indian Council
of Medical Research. The conduct of the study, laboratory analyses, data analyses
and write up were performed exclusively by the research team.